Support is requested for an Fellowship (F32) interdisciplinary project involving Physicists and Vascular Cell Biologists. It is proposed to develop and optimize an experimental platform and novel microfluidic devices to enable high-throughput analyses of responses of endothelial cells to different levels of steady and varying hydrodynamic shear by creating "shear arrays." The microfluidic devices will be used in combination with genetically-encoded biosensors and advanced fluorescence microscopy to visualize signaling events in living endothelial cells in response to shear stresses in real time with unprecedented spatial and temporal resolution. Specifically, the applicant will design and fabricate microfluidic devices made of transparent silicone elastomer polydimethylsiloxane (PDMS) that are fully compatible with real-time high-resolution brightfield and fluorescence microscopy. These devices will be adapted for cultured endothelial cells and iteratively optimized for (1) exposure of endothelial cells to shear flows of different magnitudes, covering a wide range of shear stresses for high-throughput real-time tests of response to shear;(2) generation of unidirectional pulsatile flows with well-defined amplitude and time pattern of shear stress to visualize rapid and long-term responses of cells to changes in shear stress;(3) generation of flows with the direction changing in time to study responses of cells to changes in the direction of shear stress. As a proof of principal, the applicant will test the hypothesis that the activity of endothelial cAMP-dependent protein kinase (PKA) is regulated by shear stress. A membrane-targeted genetically-encoded Forster Resonance Energy Transfer (FRET) probe will be used to visualize changes in membrane-associated PKA activity in endothelial cells subjected to varying shear stress. Relevance to public health: Alterations of shear stress in blood flow are sensed by endothelial cells and result in regulation of vascular tone, vascular permeability, and vascular remodeling. These exploratory studies have the potential to enable the facile analysis of flow-mediated signaling in living endothelial cells.